Fig. 2. The C-terminus of Sun1 associates directly with the luminal domains of Nesprin-1 and Nesprin-2. (A) Domain organisation of mouse Sun1. The domain locations as well as their amino acid positions are indicated according to the GenBank entry AAH48156 CC, coiled-coil domain; ZnF, zinc-finger domain; Tm, transmembrane domain. (B) Sun1 polypeptides corresponding to various Sun1 domains were fused to the Gal4 activating domain, whereas the Nesprin-1 luminal domain was fused to the Gal4 DNA-binding domain. The corresponding plasmids were co-transformed into yeast cells and the interactions were assessed by the filter lift ß-galactosidase assay. ++++, strong; ++, weak; –, no blue colour development. (C) Schematic overview of the fusion proteins (GST-LDN-1, GST-LDN-2 and GST-LDN-2P lacking the last 4 aa) used for the GST pull-down assay of COS7 cell homogenates expressing GFP-Sun1-C. LDN-1, luminal domain Nesprin-1; LDN-2, luminal domain Nesprin-2. (D,E) COS7 cell lysates expressing the C-terminal half of Sun1 (Sun1-C) were incubated with the immobilised GST-fusion proteins as indicated and GST for control. Unbound (S) and specifically bound (P) proteins were subjected to SDS-PAGE followed by western blot analysis using GFP-specific mAb K3-184-2.